Development of CRISPR/Cas9 edited Cryptosporidium parvum as a live attenuated vaccine candidate

Abstract

Cryptosporidium spp is a waterborne protozoan parasite that leads to diarrheal disease in humans. Despite high morbidity and mortality in millions of young children and immunocompromised patients each year, there is no effective treatment available. A drug targeting a novel Cryptosporidium parvum gene has previously been demonstrated to rescue susceptible IFNγ receptor knockout (IFNγR-KO) mice to otherwise lethal doses of C. parvum. Using CRISPR/Cas9 gene editing, this research project seeks to create a knockout C. parvum parasite strain to investigate its potential efficacy as a live-attenuated vaccine. It is hypothesized that pre-exposure of IFNγR-KO mice to the knockout C. parvum strain will elicit robust mucosal immunity that will reduce parasite burden when challenged with the parental strain. Thus far, constructs for the CRISPR/Cas9 reaction and optimization of transfection conditions have been completed. Currently, we are working on cultivating a stock of the knockout C. parvum strain. Following this, a mouse study will be conducted by orally administering transgenic parasite in IFNγR-KO mice and challenging them with the parental strain a few weeks later. Effectiveness in reducing parasite burden will be measured by C. parvum oocyst quantification in the feces and intestines through flow cytometry and qPCR. Additionally, serum, intestines and mesenteric lymph nodes will be collected before and after challenge to investigate production of C. parvum specific antibodies (i.e. IgG, IgA, and IgM), and memory cells using ELISA and flow cytometry. This preliminary mouse study with our attenuated parasite will be a step towards finding a novel vaccine for cryptosporidiosis.

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